DNA, DNA constructs, cells and plants derived therefrom

ABSTRACT

DNA constructs comprise a DNA sequence homologous to some or all of the pectinesterase gene encoded by the clone pB8 (Sequence ID No.1), under control of a transcriptional initiation region operative in plants for transcribing this DNA sequence, optionally in the antisense direction to produce RNA complementary to the gene mRNA. From such constructs may be derived transformed plant cells and plants in which expression of pectinesterase genes is inhibited: fruit from the plants (such as tomatoes) can show modified ripening properties.

This invention relates to novel DNA constructs, plant cells containing the constructs and plants derived therefrom. In particular it involves the use of antisense or sense RNA technology to control gene expression in plants.

During tomato fruit ripening, the components of the cell walls undergo a series of modifications. The pectin component is subject so substantial modification and degradation. The major changes to the pectin include: solubilisation, depolymerisation and demethylation. The changes in texture of the fruit that occur during ripening occur in parallel with the changes to the pectin component. It has been demonstrated that many of these changes are the result of the action of enzymes in the cell wall.

The cell wall enzymes polygalacturonase and pectin esterase have a major role in modifying pectin during fruit ripening. Individual cDNA clones that corresponded to mRNAs encoding these two enzymes have previously been identified and characterised. DNA sequences from these clones have been used to demonstrate sense and antisense inhibition of these enzymes (EPA 271,988, ICI). For antisense inhibition of, say, pectin esterase, a fragment of DNA coding for the mRNA that produces pectinesterase is inserted into the plant genome under control of a plant promoter, but in reverse orientation. In this way the plant produces RNA sequences complementary to the mRNA that is translated into pectin esterase: and this interferes in some way with the translation of the mRNA into protein. For sense inhibition of, say, polygalacturonase, a fragment of DNA coding for the mRNA that produces polygalacturonase is inserted into the plant genome under control of a plant promoter, in normal orientation. The plant produces RNA sequences homologous with part of the full-length mRNA that is translated into polygalacturonase. Again, this interferes with and can substantially inhibit the translation of polygalacturonase mRNA into protein. How this happens is not clear.

In this invention, we provide clones comprising further novel DNA coding sequences from a new pectin esterase gene. These can be used to make DNA constructs that will control expression of pectinesterase, either by sense or antisense inhibition.

In work leading to the invention, we have identified a novel cDNA (pB8) which encodes a tomato pectin esterase isoenzyme. The cDNA was identified in a clone from a library constructed using mRNA isolated from tomatoes at an early ripening stage. The cDNA in the clone was approximately 2 kb long. Characterisation of the sequence of the clone indicated in addition to sequences similar to those found in the previously characterised cDNA sequence (pPE1), there was also a 358 bp extension at the 5' end. This extended sequence was not predicted since the size of the previous cDNA (pPE1-1.655 kb) corresponded to the determined size of the mRNA (1.6 kb). More recent, unpublished determinations indicate that the true size of the PE mRNA in tomato fruit is approximately 2.0 kb (and see Handa, 1992).

According to the present invention, we further provide recombinant DNA comprising an upstream promoter base sequence, a base sequence for transcription into mRNA under control of said upstream promoter base sequence, and a downstream transcription terminator base sequence, characterised in that the base sequence for transcription comprises a sequence of bases complementary to a substantial run of bases in the novel PE cDNA sequence. The sequence for transcription may be oriented in the normal or reverse sense, so as to produce either sense or antisense RNA in plants. The invention also includes plant cells containing such constructs; plants derived therefrom showing modified ripening characteristics; seeds of such plants; and novel recombinant DNA from clones B8 and B16.

DNA constructs according to the invention comprise a base sequence for transcription at least 10 bases in length. For antisense transcription at least, there is no theoretical upper limit to the base sequence--it may be as long as the relevant mRNA produced by the cell--but for convenience it will generally be found suitable to use sequences between 100 and 1000 bases in length. For sense transcription, it may be found that sequences approaching the full length of the corresponding mRNA may lead to over-production of the enzyme rather than inhibition. Where this happens shorter sequences should be used, unless over-production is desired. The preparation of suitable constructs is described in more detail below.

The preferred DNA for use in the present invention is DNA derived from the clone B8. The required antisense DNA can be obtained in several ways: by cutting with restriction enzymes an appropriate sequence of such DNA; by synthesising a DNA fragment using synthetic oligonucleotides which are annealed and then ligated together in such a way as to give suitable restriction sites at each end; by using synthetic oligonucleotides in a polymerase chain reaction (PCR) to generate the required fragment with suitable restriction sites at each end. The DNA is then cloned into a vector containing upstream promoter and downstream terminator sequences, the cloning being carried out so that the cut DNA sequence is inverted with respect to its orientation in the strand from which it was cut.

In new vectors expressing antisense RNA, the strand that was formerly the template strand becomes the coding strand, and vice versa. The new vector will thus encode RNA in a base sequence which is complementary to the sequence of pB8 mRNA. Thus the two RNA strands are complementary not only in their base sequence but also in their orientations (5' to 3').

As source of the DNA base sequence for transcription, it is convenient to use cDNA clones such as pB8. The base sequences of pB8 are set out in Table 1. pB8 has been deposited on 06 Dec. 1991 with the National Collections of Industrial and Marine Bacteria, Aberdeen, under Accession No. NCIB 40463. Alternatively, cDNA clones similar to pB8 may be obtained from the mRNA of ripening tomatoes by the methods similar to that described by Slater et al, Plant Molecular Biology 5, 137-147, 1985. In this way may be obtained sequences coding for the whole, or substantially the whole, of the mRNA produced by pB8. Suitable lengths of the cDNA so obtained may be cut out for use by means of restriction enzymes.

As previously stated, DNA suitable for use in the present invention is DNA showing homology to the gene encoded by the clone pB8. pB8 was derived from a cDNA library isolated from early ripening tomato RNA.

An alternative source of DNA for the base sequence for transcription is a suitable gene encoding the pectinesterase mRNA represented in pB8. Such a gene may be isolated, for example, by probing tomato DNA with cDNA from clone pB8. This gene may differ from the cDNA of, e.g. pB8 in that introns may be present. The introns are not transcribed into mRNA (or, if so transcribed, are subsequently cut out). When using such a gene as the source of the base sequence for transcription it is possible to use either intron or exon regions.

A further way of obtaining a suitable DNA base sequence for transcription is to synthesise it ab initio from the appropriate bases, for example using Table 1 as a guide.

Recombinant DNA and vectors according to the present invention may be made as follows. A suitable vector containing the desired base sequence for transcription (for example pB8) is treated with restriction enzymes to cut the sequence out. The DNA strand so obtained is cloned desired, in reverse orientation) into a second vector containing the desired promoter sequence (for example cauliflower mosaic virus 35S RNA promoter or the tomato polygalacturonase gene promoter sequence--UK Patent Application 9024323.9) and the desired terminator sequence (for example the 3' of the Agrobacterium tumefaciens nopaline synthase gene, the nos 3' end).

According to the invention we propose to use both constitutive promoters (such as cauliflower mosaic virus 35S RNA) and inducible or developmentally regulated promoters (such as the ripe-fruit-specific polygalacturonase promoter) as circumstances require. Use of a constitutive promoter will tend to affect functions in all parts of the plant: while by using a tissue-specific promoter, functions may be controlled more selectively. Thus in applying the invention, e.g. to tomatoes, it may be found convenient to use the promoter of the PG gene (UK Patent Application 9024323.9, filed 8 Nov. 1990). Use of this promoter, at least in tomatoes, has the advantage that the production of antisense RNA is under the control of a ripening-specific promoter. Thus the antisense RNA is only produced in the organ in which its action is required. Other ripening-specific promoters that could be used include the E8 promoter (Diekman & Fischer, 1988 cited above). It may be advantageous to use a ripening-specific promoter that switches on in unripe fruit, so as to leinhibit the formation of pectinesterase from then on.

Vectors according to the invention may be used to transform plants as desired, to make plants according to the invention. Dicotyledonous plants, such as tomato and melon, may be transformed by Agrobacterium Ti plasmid technology, for example as described in EP 271,988 (ICI). Such transformed plants may be reproduced sexually, or by cell or tissue culture.

The degree of production of sense or antisense RNA in the plant cells can be controlled by suitable choice of promoter sequences, or by selecting the number of copies, or the site of integration, of the DNA sequences according to the invention that are introduced into the plant genome. In this way it may be possible to modify ripening or senescence to a greater or lesser extent.

The constructs of our invention may be used to transform cells of both monocotyledonous and dicotyledonous plants in various ways known to the art. In many cases such plant cells (particularly when they are cells of dicotyledonous plants) may be cultured to regenerate whole plants which subsequently reproduce to give successive generations of genetically modified plants. Such plants have modified fruit ripening properties. Examples of genetically modified plants according to the present invention include, as well as tomatoes, fruits of such plants as mangoes, peaches, apples, pears, strawberries, bananas and melons.

Tomatoes with reduced pectin esterase activity give improved characteristics in paste made from the processed tomatoes: in particular increased viscosity of the serum from the paste and decreased granularity of the paste, which eventually gives a smoother and more glossy paste.

The invention will now be described further with reference to Table 1, which shows the full base sequence of the clone pB8 (SEQ ID No:1) and, for comparison, the base sequence of the clone pB16 (SEQ ID No:2), homologous to the pectinesterase gene disclosed in EPA 271,988.

The following Examples illustrate aspects of the invention.

EXAMPLE 1 Identification and Characterisation of Novel PE cDNA

A cDNA library in lambda zapII (Stratagene) which had been prepared from mRNA extracted from early ripening tomato fruit was screened with a radiolabelled oligonucleotide probe corresponding to a sequence from the 5' end of pPE1 (Ray et al 1988). Several hybridising clones were identified and plasmid DNA was excised in vivo according to the instructions supplied by Stratagene. The sizes of the cDNA inserts in the clones were determined by digestion with BamH1 and KpnI followed by agarose gel electrophoresis. One clone (pB8) had an insert size of approximately 2 kb. This clone was selected for further analysis since the insert was approximately 300 bases longer than that of the previously identified PE cDNA (pPE1). The nucleotide sequence of the 5' 530 bases of this clone were determined. The 5' end of pPE1 was located at base 359 of the new clone.

EXAMPLE 2 Construction of a Constitutively Expressed PE Antisense Vector

A plant transformation vector is constructed using the sequences corresponding to bases 1 to 338 of pB8 (FIG. 1). This fragment is synthesised by polymerase chain reaction using synthetic primers. The fragment is cloned into the vector pJR1 which is previously cut with SmaI. pJR1 (Smith et al Nature 334, 724-726, 1988) is a vector hased on Bin19 (Bevan, Nucleic Acids Research, 12, 8711-8721, 1984), which permits the expression of the antisense RNA under the control of the CaMV 35S promoter. This vector includes a nopaline synthase (nos) 3' end termination sequence.

After synthesis of the vector, the structure and orientation of the PE sequences is confirmed by DNA sequence analysis.

EXAMPLE 3 Construction of a Developmentally Regulated PE Antisense Vector

The fragment of the pB8 cDNA that was described in example 2 is also cloned into the vector pJR3.

pJR3 is a Bin19-based vector, which permits the expression of the antisense RNA under the control of the tomato polygalacturonase promoter. This vector includes approximately 5 kb of promoter sequence and 1.8 kb of 3' sequence from the PG promoter separated by a multiple cloning site.

After synthesis, vectors with the correct orientation of pB8 sequences are identified by DNA sequence analysis.

EXAMPLE 4 Construction of a Constitutively Expressed PE Sense Vector

The fragment of pB8 cDNA that was described in example 2 is also cloned into the vector pJR1 in the sense orientation.

After synthesis, the vectors with the sense orientation of pB8 sequence are identified by DNA sequence analysis.

EXAMPLE 5

Construction of a Developmentally Regulated PE Sense Vector

The fragment of pB8 cDNA that was described in example 2 is also cloned into the vector pJR3 in the sense orientation.

After synthesis, the vectors with the sense orientation of pB8 sequence are identified by DNA sequence analysis.

EXAMPLE 6 Production of Genetically Modified Tomato Plants

Vectors are transferred to Agrobacterium tumefaciens LBA4404 (a micro-organism widely available to plant biotechnologists) and are used to transform tomato plants. Transformation of tomato stem segments follow standard protocols (e.g. Bird et al Plant Molecular Biology 11, 651-662, 1988--leaf or cotyledon segments may also be used). Transformed plants are identified by their ability to grow on media containing the antibiotic kanamycin. Plants are regenerated and grown to maturity. Ripening fruit are analysed for reduced PE activity and improved fruit quality characteristics.

                                      TABLE 1     __________________________________________________________________________     SEQ ID NO:   1     SEQUENCE TYPE:                  Nucleotide     SEQUENCE LENGTH:                  1989     STRANDEDNESS:                  Single     TOPOLOGY:    Linear     MOLECULE TYPE:                  DNA     ORIGINAL SOURCE ORGANISM:                            Tomato var Ailsa Craig     IMMEDIATE EXPERIMENTAL SOURCE:                            cDNA clone B8     FEATURES:     From 16 to 1654              Open reading frame encoding Pectin Esterase     From 16 to 703              Coding region for N-terminal extension     PROPERTIES:              CDNA for tomato fruit pectin esterase     TTGCAAACTT               CTAAAATGGC                         TAATCCTCAA                                   CAACCTTTCT                                             TAATAAAAAC                                                       ACACAAACAA                                                                 60     AATCCAATAA               TCAGCTTCAA                         GATCCTCAGT                                   TTTGTTATAA                                             CTTTGTTTGT                                                       TGCTCTCTTC                                                                 120     TTAGTTGCTC               CATATCAAGT                         TGAGATTAAA                                   CATTCTAATC                                             TATGTAAAAC                                                       TGCACAAGAT                                                                 180     TCCCAACTCT               GTCTCAGTTA                         TGTCTCTGAT                                   TTGATATCCA                                             ATGAAATTGT                                                       CACAACAGAA                                                                 240     TCAGATGGAC               ATAGTATTCT                         GATGAAATTT                                   TTAGTTAACT                                             ATGTCCATCA                                                       AATGAACAAT                                                                 300     GCAATTCCAG               TGGTTCGCAA                         AATGAAGAAT                                   CAGATCAATG                                             ACATTCGTCA                                                       ACACGGGGCT                                                                 360     TTAACTGATT               GTCTTGAGCT                         TCTTGATCAG                                   TCAGTTGATT                                             TCGCATCTGA                                                       TTCAATTGCA                                                                 420     GCAATTGATA               AAAGAAGTCG                         CTCGGAGCAT                                   GCCAATGCGC                                             AAAGTTGGCT                                                       AAGTGGTGTG                                                                 480     CTTACTAACC               ACGTTACGTG                         CTTGGATGAG                                   CTTGATTCCT                                             TTACTAAAGC                                                       TATGATAAAT                                                                 540     GGAACGAATC               TTGAAGAGTT                         GATCTCGAGA                                   GCTAAGGTAG                                             CATTAGCGAT                                                       GCTTGCGTCT                                                                 600     TTGACAACTC               AGGATGAGGA                         TGTTTTCATG                                   ACGGTTTTAG                                             GAAAAATGCC                                                       ATCTTGGGTG                                                                 660     AGTTCGATGG               ATAGGAAGCT                         GATGGAGAGT                                   TCGGGTAAGG                                             ACATTATAGC                                                       GAATGCAGTG                                                                 720     GTGGCACAAG               ATGGAACGGG                         GGATTATCAA                                   ACACTTGCTG                                             AAGCAGTTGC                                                       TGCAGCACCA                                                                 780     GATAAGAGTA               AGACGCGTTA                         TGTAATTTAT                                   GTAAAGAGGG                                             GAACTTATAA                                                       AGAGAATGTT                                                                 840     GAGGTGGCTA               GCAATAAAAT                         GAACTTGATG                                   ATTGTTGGTG                                             ATGGAATGTA                                                       TGCTACGACC                                                                 900     ATTACTGGTA               GCCTTAATGT                         TGTCGATGGA                                   TCAACAACCT                                             TCCGCTCTGC                                                       CACTCTTGCT                                                                 960     GCAGTCGGCC               AAGCATTTAT                         ACTACAGGAC                                   ATATGTATAC                                             AGAACACAGC                                                       AGGGCCAGCG                                                                 1020     AAAGACCAAG               CAGTGGCACT                         TCGAGTTGGA                                   GCTGATATGT                                             CTGTCATAAA                                                       TCGTTGTCGT                                                                 1080     ATCGATGCTT               ATCAAGACAC                         CCTTTATGCA                                   CATTCTCAAA                                             GGCAATTCTA                                                       TCGAGACTCC                                                                 1140     TACGTGACAG               GTACTGTTGA                         TTTCATATTT                                   GGTAATGCAG                                             CAGTTGTATT                                                       CCAGAAATGC                                                                 1200     CAGCTCGTAG               CTAGAAAACC                         GGGTAAATAC                                   CAGCAAAACA                                             TGGTGACTGC                                                       ACAAGGCAGG                                                                 1260     ACGGACCCAA               ATCAGGCCAC                         GGGGACATCA                                   ATTCAGTTCT                                             GTAACATAAT                                                       AGCAAGTTCG                                                                 1320     GACCTAGAAC               CAGTCCTGAA                         AGAATTCCCA                                   ACATATCTTG                                             GTAGGCCATG                                                       GKAAGAATAT                                                                 1380     TCAAGAACTG               TAGTGATGGA                         ATCATACTTA                                   GGTGGTCTCA                                             TTAATCCAGC                                                       GGGTTGGGCT                                                                 1440     GAGTGGGACG               GAGATTTTGC                         GTTGAAGACA                                   TTGTATTATG                                             GTGAATTTAT                                                       GAACAATGGA                                                                 1500     CCTGGTGCTG               GTACTAGTAA                         GCGTGTCAAG                                   TGGCCTGGTT                                             ATCATGTCAT                                                       TACTGATCCC                                                                 1560     GCTAAAGCTA               TGCCGTTCAC                         TGTGGCTAAG                                   CTGATTCAGG                                             GCGGATCATG                                                       GTTGAGGTCT                                                                 1620     ACTGGCGTGG               CGTATGTGGA                         TGGATTATAT                                   GATTAGAGTA                                             TATATATGAT                                                       GTGCCACATG                                                                 1680     AGCAGGGCAG               AGCAAGCATA                         ACACACAACT                                   CTAGTGTGAC                                             AAGCATTTAC                                                       ATGGCTCATT                                                                 1740     CGTTACTACT               AAGTTGTCAA                         TAAGTTCTGT                                   TTAGGGGTTC                                             ATAAGTTTAT                                                       ATACGTATAT                                                                 1800     ACATTTACAT               TGGTGATGAA                         GCTGAAACTG                                   ATGATGCTTT                                             AATGTAATTA                                                       TAGTTTTCTG                                                                 1860     AAAAAGGATA               TGAGTAATAT                         TAGTTTTTCC                                   CAGATGTGTA                                             TGGTTGTGGA                                                       ACTGTTTATG                                                                 1920     CTTAAATTGG               CAAGGGGTAT                         TGAATAAAAA                                   TCTATTGTGT                                             TAAAAAAAAA                                                       AAAAAAAAAA                                                                 1980     AAAAAAAAAA                                                  1989     __________________________________________________________________________

                                      TABLE 2     __________________________________________________________________________     SEQ ID NO:   2     SEQUENCE TYPE:                  Nucleotide     SEQUENCE LENGTH:                  2059     STRANDEDNESS:                  Single     TOPOLOGY:    Linear     MOLECULE TYPE:                  DNA     ORIGINAL SOURCE ORGANISM: Tomato var Ailsa Craig     IMMEDIATE EXPERIMENTAL SOURCE: cDNA clone B16     FEATURES:     From 13 to 1672              Open reading frame encoding Pectin Esterase     From 13 to 711              Coding region for N-terminal extension     Base 368 Start of cDNA pPE1 published by Ray et al (1988)     PROPERTIES:              cDNA for tomato fruit pectin esterase     CGAACTTCTA               AAATGGCTAC                         TCCTCAACAA                                   CCTTTGTTAA                                             CAAAAACACA                                                       CAAACAAAAT                                                                 60     TCCATAATCA               GCTTCAAGAT                         CCTCACTTTT                                   GTTGTAACTT                                             TGTTTGTTGC                                                       TCTCTTCTTA                                                                 120     GTTGTGTTTC               TTGTTGCTCC                         ATATCAATTT                                   GAGATTAAAC                                             ATTCTAATCT                                                       GTGTAAAACT                                                                 180     GCACAAGATT               CCCAACTCTG                         TCTCAGTTAT                                   GTTTCTGATT                                             TAATATCCAA                                                       TGAAATTGTC                                                                 240     ACATCTGATT               CAGATGGACT                         AAGTATTCTG                                   AAGAAATTTT                                             TAGTTTACTC                                                       TGTTCATCAA                                                                 300     ATGAACAATG               CAATTCCAGT                         GGTTCGCAAA                                   ATCAAGAATC                                             AGATCAATGA                                                       CATTCGTGAA                                                                 360     CAAGGGGCTT               TAACTGATTG                         TCTTGAGCTT                                   CTTGATCTGT                                             CAGTTGATTT                                                       AGTATGTGAT                                                                 420     TCAATTGCAG               CAATTGATAA                         GAGAAGTCGT                                   TCGGAGCATG                                             CCAATGCGCA                                                       AAGTTGGCTA                                                                 480     AGTGGTGTGC               TTACTAACCA                         CGTTACGTGC                                   TTGGATGAGC                                             TTGATTCCTT                                                       TACTAAAGCT                                                                 540     ATGATAAATG               GAACGAATCT                         TGATGAGTTG                                   ATCTCGAGAG                                             CTAAGGTAGC                                                       ATTGGCGATG                                                                 600     CTTGCGTCTG               TGACAACTCC                         AAATGATGAA                                   GTTTTGAGGC                                             CGGGTTTAGG                                                       AAAAATGCCA                                                                 660     TCTTGGGTGA               GTTCGAGGGA                         TAGGAAGCTG                                   ATGGAGAGTT                                             CGGGTAAGGA                                                       CATTGGAGCG                                                                 720     AATGCAGTGG               TGGCAAAAGA                         TGGAACAGGG                                   AAATATCGAA                                             CACTTGCTGA                                                       AGCTGTTGCT                                                                 780     GCAGCACCAG               ATAAGAGTAA                         GACGCGTTAT                                   GTAATTTATG                                             TAAAGAGGGG                                                       AACTTATAAA                                                                 840     GAGAATGTTG               AGGTGAGTAG                         CAGGAAAATG                                   AATTTGATGA                                             TTATTGGTGA                                                       TGGCATGTAT                                                                 900     GCTACCATCA               TTACTGGGAG                         CCTTAATGTT                                   GTCGATGGAT                                             CAACAACCTT                                                       CCACTCTGCC                                                                 960     ACTCTTGCTG               CAGTTGGCAA                         AGGATTTATA                                   CTACAGGACA                                             TATGTATACA                                                       GAACACAGCA                                                                 1020     GGACCAGCTA               AACACCAAGC                         TGTTGCACTT                                   CGAGTTGGAG                                             CTGATAAGTC                                                       TGTCATAAAT                                                                 1080     CGTTGTCGTA               TCGATGCTTA                         TCAAGACACC                                   CTTTATGCAC                                             ATTCTCAAAG                                                       GCAATTCTAT                                                                 1140     CGAGACTCCT               ACGTGACAGG                         GACTATTGAT                                   TTCATATTCG                                             GTAATGCAGC                                                       AGTTGTATTC                                                                 1200     CAGAAATGCC               AGCTCGTAGC                         TAGAAAACCG                                   GGTAAATACC                                             AGCAAAACAT                                                       GGTGACTGCA                                                                 1260     CAAGGCAGGA               CGGACCCAAA                         TCAGGCCACG                                   GGGACATCAA                                             TTCAGTTTTG                                                       TGATATAATA                                                                 1320     GCAAGTCCTG               ACCTAAAACC                         AGTCGTGAAA                                   GAATTCCCAA                                             CATATCTTGG                                                       TAGGCCATGG                                                                 1380     AAAAAATATT               CAAGAACTGT                         AGTGATGGAA                                   TCATCATTGG                                             GTGGTCTCAT                                                       TGATCCATCG                                                                 1440     GGTTGGGCTG               AGTGGCACGG                         AGATTTTGCG                                   TTAAAGACAT                                             TGTATTATGG                                                       TGAATTTATG                                                                 1500     AATAATGGAC               CTGGTGCTGG                         TACTAGTAAG                                   CGTGTCAAGT                                             GGCCTGGCTA                                                       TCATGTCATT                                                                 1560     ACTGACCCCG               CTGAAGCTAT                         GTCATTCACT                                   GTGGCTAAGC                                             TGATTCAGGG                                                       CGGATCATGG                                                                 1620     TTGAGGTCTA               CTGACGTGGC                         GTATGTGGAT                                   GGATTATATG                                             ATTAGAGTGA                                                       TATAAAATTA                                                                 1680     CTCTTTGTTT               ATGTAACAAG                         ACATCTTTAA                                   AAAGTTCAAA                                             GTAAGTAGTA                                                       GTAATATATC                                                                 1740     CATATGAAGT               GCCACATGAG                         CAGGGCAGAG                                   CCGGGATTAA                                             GTGTCTAAAG                                                       CATAACACAC                                                                 1800     AACTCTAGTG               TGACAAGCAT                         TTACATGGCT                                   CATTCCTTAC                                             TACTAAGTCG                                                       TCAATAAGTT                                                                 1860     CAGTTAAGGG               GTTCATAAGT                         TAATATACGT                                   ATATATATTT                                             ATGTTGGCGA                                                       TAAAGCTGAA                                                                 1920     ACTGATGATG               CTTTAATGTA                         ATTATAGTTT                                   TCTGAAAAAG                                             GATATGTGTA                                                       ATATTAGGTT                                                                 1980     TTCCCTGATG               TTTATGGTTG                         TGGGGTGGTG                                   GTTATGATAA                                             AAATATGCAA                                                       GATGAAAGTC                                                                 2040     AAAAAAAAAA               AAAAAAAAA                                         2059     __________________________________________________________________________

    __________________________________________________________________________     SEQUENCE LISTING     (1) GENERAL INFORMATION:     (iii) NUMBER OF SEQUENCES: 2     (2) INFORMATION FOR SEQ ID NO:1:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1990 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:     TTGCAAACTTCTAAAATGGCTAATCCTCAACAACCTTTGTTAATAAAAACACACAAACAA60     AATCCAATAATCAGCTTCAAGATCCTCAGTTTTGTTATAACTTTGTTTGTTGCTCTCTTC120     TTAGTTGCTCCATATCAAGTTGAGATTAAACATTCTAATCTATGTAAAACTGCACAAGAT180     TCCCAACTCTGTCTCAGTTATGTCTCTGATTTGATATCCAATGAAATTGTCACAACAGAA240     TCAGATGGACATAGTATTCTGATGAAATTTTTAGTTAACTATGTCCATCAAATGAACAAT300     GCAATTCCAGTGGTTCGCAAAATGAAGAATCAGATCAATGACATTCGTCAACACGGGGCT360     TTAACTGATTGTCTTGAGCTTCTTGATCAGTCAGTTGATTTCGCATCTGATTCAATTGCA420     GCAATTGATAAAAGAAGTCGCTCGGAGCATGCCAATGCGCAAAGTTGGCTAAGTGGTGTG480     CTTACTAACCACGTTACGTGCTTGGATGAGCTTGATTCCTTTACTAAAGCTATGATAAAT540     GGAACGAATCTTGAAGAGTTGATCTCGAGAGCTAAGGTAGCATTAGCGATGCTTGCGTCT600     TTGACAACTCAGGATGAGGATGTTTTCATGACGGTTTTAGGAAAAATGCCATCTTGGGTG660     AGTTCGATGGATAGGAAGCTGATGGAGAGTTCGGGTAAGGACATTATAGCGAATGCAGTG720     GTGGCACAAGATGGAACGGGGGATTATCAAACACTTGCTGAAGCAGTTGCTGCAGCACCA780     GATAAGAGTAAGACGCGTTATGTAATTTATGTAAAGAGGGGAACTTATAAAGAGAATGTT840     GAGGTGGCTAGCAATAAAATGAACTTGATGATTGTTGGTGATGGAATGTATGCTACGACC900     ATTACTGGTAGCCTTAATGTTGTCGATGGATCAACAACCTTCCGCTCTGCCACTCTTGCT960     GCAGTCGGCCAAGGATTTATACTACAGGACATATGTATACAGAACACAGCAGGGCCAGCG1020     AAAGACCAAGCAGTGGCACTTCGAGTTGGAGCTGATATGTCTGTCATAAATCGTTGTCGT1080     ATCGATGCTTATCAAGACACCCTTTATGCACATTCTCAAAGGCAATTCTATCGAGACTCC1140     TACGTGACAGGTACTGTTGATTTCATATTTGGTAATGCAGCAGTTGTATTCCAGAAATGC1200     CAGCTCGTAGCTAGAAAACCGGGTAAATACCAGCAAAACATGGTGACTGCACAAGGCAGG1260     ACGGACCCAAATCAGGCCACGGGGACATCAATTCAGTTCTGTAACATAATAGCAAGTTCG1320     GACCTAGAACCAGTCCTGAAAGAATTCCCAACATATCTTGGTAGGCCATGGAAAGAATAT1380     TCAAGAACTGTAGTGATGGAATCATACTTAGGTGGTCTCATTAATCCAGCGGGTTGGGCT1440     GAGTGGGACGGAGATTTTGCGTTGAAGACATTGTATTATGGTGAATTTATGAACAATGGA1500     CCTGGTGCTGGTACTAGTAAGCGTGTCAAGTGGCCTGGTTATCATGTCATTACTGATCCC1560     GCTAAAGCTATGCCGTTCACTGTGGCTAAGCTGATTCAGGGCGGATCATGGTTGAGGTCT1620     ACTGGCGTGGCGTATGTGGATGGATTATATGATTAGAGTATATATATGATGTGCCACATG1680     AGCAGGGCAGAGCAAGCATAACACACAACTCTAGTGTGACAAGCATTTACATGGCTCATT1740     CGTTACTACTAAGTTGTCAATAAGTTCTGTTTAGGGGTTCATAAGTTTATATACGTATAT1800     ACATTTACATTGGTGATGAAGCTGAAACTGATGATGCTTTAATGTAATTATAGTTTTCTG1860     AAAAAGGATATGAGTAATATTAGTTTTTCCCAGATGTGTATGGTTGTGGAACTGTTTATG1920     CTTAAATTGGCAAGGGGTATTGAATAAAAATCTATTGTGTTAAAAAAAAAAAAAAAAAAA1980     AAAAAAAAAA1990     (2) INFORMATION FOR SEQ ID NO:2:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 2059 base pairs     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: cDNA     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:     CGAACTTCTAAAATGGCTACTCCTCAACAACCTTTGTTAACAAAAACACACAAACAAAAT60     TCCATAATCAGCTTCAAGATCCTCACTTTTGTTGTAACTTTGTTTGTTGCTCTCTTCTTA120     GTTGTGTTTCTTGTTGCTCCATATCAATTTGAGATTAAACATTCTAATCTGTGTAAAACT180     GCACAAGATTCCCAACTCTGTCTCAGTTATGTTTCTGATTTAATATCCAATGAAATTGTC240     ACATCTGATTCAGATGGACTAAGTATTCTGAAGAAATTTTTAGTTTACTCTGTTCATCAA300     ATGAACAATGCAATTCCAGTGGTTCGCAAAATCAAGAATCAGATCAATGACATTCGTGAA360     CAAGGGGCTTTAACTGATTGTCTTGAGCTTCTTGATCTGTCAGTTGATTTAGTATGTGAT420     TCAATTGCAGCAATTGATAAGAGAAGTCGTTCGGAGCATGCCAATGCGCAAAGTTGGCTA480     AGTGGTGTGCTTACTAACCACGTTACGTGCTTGGATGAGCTTGATTCCTTTACTAAAGCT540     ATGATAAATGGAACGAATCTTGATGAGTTGATCTCGAGAGCTAAGGTAGCATTGGCGATG600     CTTGCGTCTGTGACAACTCCAAATGATGAAGTTTTGAGGCCGGGTTTAGGAAAAATGCCA660     TCTTGGGTGAGTTCGAGGGATAGGAAGCTGATGGAGAGTTCGGGTAAGGACATTGGAGCG720     AATGCAGTGGTGGCAAAAGATGGAACAGGGAAATATCGAACACTTGCTGAAGCTGTTGCT780     GCAGCACCAGATAAGAGTAAGACGCGTTATGTAATTTATGTAAAGAGGGGAACTTATAAA840     GAGAATGTTGAGGTGAGTAGCAGGAAAATGAATTTGATGATTATTGGTGATGGCATGTAT900     GCTACCATCATTACTGGGAGCCTTAATGTTGTCGATGGATCAACAACCTTCCACTCTGCC960     ACTCTTGCTGCAGTTGGCAAAGGATTTATACTACAGGACATATGTATACAGAACACAGCA1020     GGACCAGCTAAACACCAAGCTGTTGCACTTCGAGTTGGAGCTGATAAGTCTGTCATAAAT1080     CGTTGTCGTATCGATGCTTATCAAGACACCCTTTATGCACATTCTCAAAGGCAATTCTAT1140     CGAGACTCCTACGTGACAGGGACTATTGATTTCATATTCGGTAATGCAGCAGTTGTATTC1200     CAGAAATGCCAGCTCGTAGCTAGAAAACCGGGTAAATACCAGCAAAACATGGTGACTGCA1260     CAAGGCAGGACGGACCCAAATCAGGCCACGGGGACATCAATTCAGTTTTGTGATATAATA1320     GCAAGTCCTGACCTAAAACCAGTCGTGAAAGAATTCCCAACATATCTTGGTAGGCCATGG1380     AAAAAATATTCAAGAACTGTAGTGATGGAATCATCATTGGGTGGTCTCATTGATCCATCG1440     GGTTGGGCTGAGTGGCACGGAGATTTTGCGTTAAAGACATTGTATTATGGTGAATTTATG1500     AATAATGGACCTGGTGCTGGTACTAGTAAGCGTGTCAAGTGGCCTGGCTATCATGTCATT1560     ACTGACCCCGCTGAAGCTATGTCATTCACTGTGGCTAAGCTGATTCAGGGCGGATCATGG1620     TTGAGGTCTACTGACGTGGCGTATGTGGATGGATTATATGATTAGAGTGATATAAAATTA1680     CTCTTTGTTTATGTAACAAGACATCTTTAAAAAGTTCAAAGTAAGTAGTAGTAATATATC1740     CATATGAAGTGCCACATGAGCAGGGCAGAGCCGGGATTAAGTGTCTAAAGCATAACACAC1800     AACTCTAGTGTGACAAGCATTTACATGGCTCATTCCTTACTACTAAGTCGTCAATAAGTT1860     CAGTTAAGGGGTTCATAAGTTAATATACGTATATATATTTATGTTGGCGATAAAGCTGAA1920     ACTGATGATGCTTTAATGTAATTATAGTTTTCTGAAAAAGGATATGTGTAATATTAGGTT1980     TTCCCTGATGTTTATGGTTGTGGGGTGGTGGTTATGATAAAAATATGCAAGATGAAAGTC2040     AAAAAAAAAAAAAAAAAAA2059     __________________________________________________________________________ 

We claim:
 1. A DNA construct comprising a DNA sequence of the gene encoded by the clone pB8 (SEQ ID NO:1) under the control of a transcriptional initiation region in plants, so that the construct can generate RNA in plant cells.
 2. The DNA construct as claimed in claim 1 comprising a transcriptional initiation region operative in plants positioned for transcription of a DNA sequence encoding RNA complementary to a sequence of bases of mRNA of the gene encoded by the clone pB8 (SEQ ID NO:1).
 3. The DNA construct as claimed in claim 1 in which the transcriptional initiation region operative in plants is a constitutive promoter.
 4. The DNA construct as claimed in claim 3 in which the constitutive promoter is CaMV35S.
 5. The DNA construct as claimed in claim 1 in which the transcriptional initiation region operative in plants is an inducible or developmentally regulated promoter.
 6. The DNA construct as claimed in claim 5 in which the promoter is that for the polygalacturonase gene.
 7. Plant cells transformed with a construct claimed in claim
 1. 8. Plant cells claimed in claim 7 which are cells of tomato.
 9. Plant cells claimed in claim 7 which are cells of mangoes, peaches, apples, pears, bananas, melons or strawberries.
 10. Plants containing cells claimed in claim
 7. 11. Plants claimed in claim 10 which bear climacteric fruit.
 12. Fruit or seeds of plants claimed in claim
 11. 13. Tomato seeds as claimed in claim 12 containing a construct adapted to express RNA antisense to mRNA expressed by the gene encoded by the clone pB8 (Sequence ID No 1).
 14. Recombinant DNA comprising SEQ ID NO:1.
 15. Recombinant DNA comprising a base sequence at least 16 bases in length that is identical to the sequence from base 16 to base 1654 shown in SEQ ID NO:1 or to the sequence from base 13 to base 367 shown in SEQ ID NO:2.
 16. A DNA construct comprising a DNA sequence of clone pB8 (SEQ ID NO:1), or a gene encoding the same amino acid sequence as said DNA sequence, under the control of a transcriptional initiation region in plants, so that the construct can generate RNA in plant cells. 